BACKGROUND: The increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We aimed to explore Treponema pallidum subspecies pallidum (TPA) molecular epidemiology essential for vaccine research by analysing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data. METHODS: In this multicentre, cross-sectional, molecular epidemiology study, we enrolled patients with primary, secondary, or early latent syphilis from clinics in China, Colombia, Malawi, and the USA between Nov 28, 2019, and May 27, 2022. Participants aged 18 years or older with laboratory confirmation of syphilis by direct detection methods or serological testing, or both, were included. Patients were excluded from enrolment if they were unwilling or unable to give informed consent, did not understand the study purpose or nature of their participation, or received antibiotics active against syphilis in the past 30 days. TPA detection and WGS were conducted on lesion swabs, skin biopsies, skin scrapings, whole blood, or rabbit-passaged isolates. We compared our WGS data to publicly available genomes and analysed TPA populations to identify mutations associated with lineage and geography. FINDINGS: We screened 2802 patients and enrolled 233 participants, of whom 77 (33%) had primary syphilis, 154 (66%) had secondary syphilis, and two (1%) had early latent syphilis. The median age of participants was 28 years (IQR 22-35); 154 (66%) participants were cisgender men, 77 (33%) were cisgender women, and two (1%) were transgender women. Of the cisgender men, 66 (43%) identified as gay, bisexual, or other sexuality. Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants showed a predominance of SS14-lineage strains with geographical clustering. Phylogenomic analyses confirmed that Nichols-lineage strains were more genetically diverse than SS14-lineage strains and clustered into more distinct subclades. Differences in single nucleotide variants (SNVs) were evident by TPA lineage and geography. Mapping of highly differentiated SNVs to three-dimensional protein models showed population-specific substitutions, some in outer membrane proteins (OMPs) of interest. INTERPRETATION: Our study substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains is vital for vaccine development and understanding syphilis pathogenesis on a population level. FUNDING: US National Institutes of Health National Institute for Allergy and Infectious Disease, the Bill & Melinda Gates Foundation, Connecticut Children's, and the Czech Republic National Institute of Virology and Bacteriology.

Centro Internacional de Entrenamiento e Investigaciones Medicas Campus de la Universidad Icesi Cali Colombia

Centro Internacional de Entrenamiento e Investigaciones Medicas Campus de la Universidad Icesi Cali Colombia; Department of Pediatrics Universidad del Valle Cali Colombia

Centro Internacional de Entrenamiento e Investigaciones Medicas Campus de la Universidad Icesi Cali Colombia; Universidad Icesi Cali Colombia

Centro Internacional de Entrenamiento e Investigaciones Medicas Campus de la Universidad Icesi Cali Colombia; Universidad Icesi Cali Colombia; Division of Dermatology Department of Internal Medicine School of Medicine Universidad del Valle Cali Colombia

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Health Behavior Gillings School of Global Public Health University of North Carolina at Chapel Hill Chapel Hill North Carolina USA

Department of Medicine Division of Infectious Diseases Institute for Global Health and Infectious Diseases University of North Carolina at Chapel Hill Chapel Hill NC USA

Department of Medicine Division of Infectious Diseases Institute for Global Health and Infectious Diseases University of North Carolina at Chapel Hill Chapel Hill NC USA; Institute for Global Health and Infectious Diseases Infectious Diseases Epidemiology and Ecology Laboratory University of North Carolina at Chapel Hill Chapel Hill NC USA

Department of Medicine UConn Health Farmington CT USA

Department of Medicine UConn Health Farmington CT USA; Department of Pediatrics UConn Health Farmington CT USA; Connecticut Children's Hartford CT USA

Department of Medicine UConn Health Farmington CT USA; Department of Pediatrics UConn Health Farmington CT USA; Department of Immunology UConn Health Farmington CT USA; Connecticut Children's Hartford CT USA

Department of Pediatrics Division of Infectious Diseases Duke University Medical Center Durham NC USA; Department of Integrative Immunology Duke University Medical Center Durham NC USA; Duke Human Vaccine Institute Durham NC USA

Department of Pediatrics UConn Health Farmington CT USA; Department of Immunology UConn Health Farmington CT USA; Connecticut Children's Hartford CT USA

Dermatology Hospital Southern Medical University Guangdong Provincial Center for Skin Diseases and STD Control Guangzhou China

Dermatology Hospital Southern Medical University Guangdong Provincial Center for Skin Diseases and STD Control Guangzhou China; BSL 3 Laboratory Guangdong Provincial Key Laboratory of Tropical Disease Research School of Public Health Southern Medical University Guangzhou China

Institute for Global Health and Infectious Diseases Infectious Diseases Epidemiology and Ecology Laboratory University of North Carolina at Chapel Hill Chapel Hill NC USA

UNC Project Malawi Tidziwe Centre Lilongwe Malawi

medRxiv. 2023 Jul 24:2023.07.19.23291250. doi: 10.1101/2023.07.19.23291250. PubMed

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